Remarks on phylogeny and molecular variations of criconematid species (Nematoda: Criconematidae) with case studies from Vietnam

The family Criconematidae is a remarkable group of nematodes, containing roughly 600 nominal root-ectoparasitic species, of which many species are known to be significant agricultural pests. Strikingly, our phylogenetic analyses based on 18S, D2-D3 of 28S rRNA, and COI mtDNA sequences of criconematid species, supported by tree topology tests (SH and AU tests), revealed that almost all studied genera, including Criconema, Ogma, Crossonema, Discocriconema, Hemicriconemoides, Criconemoides, Mesocriconema, and Lobocriconema, are not monophyletic groups, a finding that is partly contrary to those of previous studies on these groups. Our results suggest that key morphological characters used in the classification of Criconematidae are the consequence of convergent evolution. It is clear from our studies that the species status of at least 40 sequences of criconematid species from GenBank must be either revised or reconsidered, with analyses based on a polyphasic approach that includes different tree- and distance-based molecular species-delimitation methods (bPTP, GMYC, ABGD1, and ABGD2). Our studies found the ABGD2 output of the automatic barcode method to agree remarkably well with established species delimitations, while in general, the four species-delimitation results corresponding to three barcode regions forwarded significantly more putative species compared to those originally considered. This study also characterised for the first time the populations of Criconemoides myungsugae and Discocriconemella hensungica associated with Vietnamese ginseng, one of the most precious and rare ginseng varieties in the world. Although these populations are morphologically in agreement with the original descriptions of C. myungsugae and D. hengsungica, their molecular data display notable variations compared to the sequences deposited in GenBank. These species demonstrate clearly the immense molecular variations that can be observed in several species of the family Criconematidae.


Materials and methods
Soil samples were collected from the rhizosphere of Vietnamese ginseng (Panax vietnamensis Ha & Grushv) in the Western Highlands, Vietnam. Nematodes were extracted using the modified Baerman tray method 13 . For morphological characterisation, nematodes were fixed in TAF for a week and transferred to glycerine to make permanent slides following the method of Seinhorst 14 . Measurements and pictures were taken from nematodes in permanent slides using a Carl Zeiss Axio Lab.A1 light microscope equipped with an Axiocam ERc5s digital camera.
For molecular analyses, a single individual of living nematodes was used for DNA extraction. Living nematodes were cut into several pieces and transferred to a PCR tube with 20 μl of WLB (50 mM KCl;10 mM Tris pH 8.3; 2.5 mM MgCl2; 0.45% NP-40 (Tergitol Sigma); 0.45% Tween -20). Subsequently, the sample was incubated at − 20 °C for 10 min, followed by adding 1 μl proteinase K (1.2 mg ml − 1), incubating in a PCR machine for 1 h at 65 °C and 10 min at 95 °C.
Monophyly of studied genera was tested using the Shimodaira-Hasegawa (1989) and the approximately unbiased tests (SH and AU tests) in IQ-TREE multicore version 2.2.0 26 . Constrained tree for each genus was created using Notepad + + v7.5.6 and the same alignments as above were used for the tests. www.nature.com/scientificreports/ breaks and anastomoses of transverse striae; first lip annulus forming an uninterrupted disc (Fig. 1b); robust and straight stylet with anchor-shaped knobs; typical pharynx of the genus, with fused procorpus and metacorpus, a large valve, a swollen and offset basal bulb from narrowed isthmus (Fig. 1b); small and oval-shaped spermatheca without sperm; monodelphic-prodelphic ovary; closed vulva, flat or slightly protruding above body contour (Fig. 1c); and broadly rounded or slightly tapering tail (Fig. 1d).

Characterisation of
Morphological relationship. According to the identification key provided by Geraert 1 , the Vietnamese Criconemoides population is most similar to Criconemoides morgensis and Criconemoides myungsugae. However, our nematode population can be clearly distinguished from Criconemoides morgensis by first annulus (disc-like vs not disc-like), spermatheca (empty vs filled with sperm), smaller body length (395-468 vs 510-700 µm), smaller stylet length (72-77 vs 74-91 µm), and other measurements (see Table 1). Besides, the morphology of our nematode population is in agreement with the original and redescription of the type population of Criconemoides myungsugae, except for the slightly longer stylet (72-77 vs 66-72 µm) and smaller c value (15.5-22 vs 21-26) 28,30 . Furthermore, our population of Criconemoides species also showed very high similarity compared to other populations of C. myungsugae from Iran, China, Switzerland and the US (Table 1). Therefore, our nematode population is identified morphologically as C. myungsugae.
Molecular characterisation and relationship. Characterisation of 18S rRNA region. Two 18S rRNA sequences of 837 bp length were obtained for C. myungsugae from Vietnam. The intraspecific variation between our sequences was 1% (6 bp difference). Our 18S rRNA sequences were 98.6-99.6% similar (5-14 bp difference) to the sequences of C. myungsugae from GenBank (accession number: MZ041014, MH444645, MH444644). Strikingly, they were also 98.8-99.3% similar (only 6-10 bp difference) to the 18S rRNA sequences of C. annulatus from GenBank (MF095008, MF095024, MF095015, MF094901). The phylogenetic tree based on 18S rRNA sequences showed that the sequence of C. myungsugae from Vietnam was embedded in a poorly-supported clade of C. myungsugae (PP 0.79), and in a maximally-supported (PP 1), clade of both C. myungsugae and C. annulatus (Fig. 2).  www.nature.com/scientificreports/ However, only one species-delimitation result (ABGD2) recognised one of our 18S rRNA sequences as being conspecific with C. myungsugae (MH444645, MH444644). Interestingly, despite the fact that our two 18S sequences were from the same population, two species-delimitation results (GMYC and ABGD1) indicated all five 18S rRNA sequences of C. myungsugae (including our sequences) to be five separated species (Fig. 2).
Characterisation of D2-D3 of 28S rRNA region. Two D2-D3 sequences of C. myungsugae from Vietnam were obtained with 3% intraspecific variation (721-742 bp long). These sequences were found to be most similar to the sequences of C. myungsugae (MZ041096, MW938533, MW938532, MH444643, MH444641, and MH444642) with only 94.9-97.7% similarity (17-34 bp difference). Despite these relatively large differences, the phylogenetic tree based on D2-D3 sequences showed that the sequences of C. myungsugae from Vietnam are imbedded in a maximally supported C. myungsugae clade, which has a sister relationship to the sequences of Criconemoides   www.nature.com/scientificreports/ Characterisation of COI mtDNA region. Two obtained COI sequences of C. myungsugae from this study were 439-440 bp long without intraspecific sequence variation. They were only 89% similar (46 bp difference) to the closest sequences of C. myungsugae (MH496163, MH496164, MH496165). The resulting COI phylogenetic tree showed that the sequences of C. myungsugae from Vietnam have a maximal supported sister relationship to the sequence of C. myungsugae from GenBank and the C. myungsugae clade has a not well-supported sister relationship to one sequence of C. annulatus (MF770893), making sequences of C. annulatus paraphyletic group (Fig. 4).
Remarkably, all four species-delimitation results indicated the two COI sequences of C. myungsugae of Vietnam as a different species as C. myungsugae from GenBank (Fig. 4).

Characterisations of Discocriconemella hengsungica Choi & Geraert, 1975 from Vietnam.
Measurements. All measurements of Discocriconemella hengsungica from Vietnam are provided in Table 2.
Characterization of D2-D3 of 28S rRNA region. The D2-D3 sequence of D. hengsungica from Vietnam was 749 bp long and most similar to of D. hengsungica (MK253536) with 96% similarity (21 bp difference). The resulting D2-D3 tree topology indicated a maximally supported relationship of all D. hengsungica sequences (Fig. 3). Despite the remarkably dissimilarity between the two D. hengsungica sequences, two ABGD speciesdelimitation results confirmed their conspecificity. However, bPTP and GMYC species-delimitation methods recognised these sequences as two different species (Fig. 3).
Characterisation of COI mtDNA region. The COI sequences of D. hengsungica obtained from this study was 359 bp long and only 88% similar (44 bp difference) to the sequence of D. hengsungica from GenBank (MF770969). Nevertheless, the aforementioned sequences were placed in a maximally supported sister relationship (Fig. 4). Similar to the case of C. myungsugae, all four species-delimitation results recognised these sequences as two separated species (Fig. 4). However, several studies indicated that the molecular variation of COI can be relatively large between different populations of a single species 27,31 . Consequently, all the speciesdelimitation methods used in this study recognised much higher number of molecular species based on COI than the number of corresponding morpho-species (Table 3).
Remarks on molecular variation and phylogenetic relationship of studied species. It is clear that phylogenetic positions of certain genera in the family Criconematidae are not well-resolved, and that almost none of the studied genera form monophyletic groups (Fig. 6). On the tree constructed from 18S rRNA sequences, virtually all genera, including Criconema, Ogma, Crossonema, Discocriconemella, Hemicriconemoides, Criconemoides, Mesocriconema, and Lobocriconema, appeared in at least two different clades (Fig. 6). No conclusions can be drawn regarding Bakernema since only the sequences of one species, Bakernema inaequale, are available (Fig. 2). Similarly, the phylogenetic trees created from D2-D3 and COI sequences also show the non-monophyly of the above genera, albeit relatively weakly-supported, since the posterior probabilities at crucial nodes are relatively low (Figs. 2, 3, 4, 6). Some genera, including Nothocriconemoides and Neolobocriconema, appear to be monophyletic; most likely because of a more limited representation of sequences compared to the 18S analyses (Figs. 3, 4).
The high molecular variations in the family Criconematidae alongside our tree-and distance-based molecular species-delimitation results and other available data suggest that 40 sequences, of 14 different species, on GenBank are required to be either corrected or re-considered ( Table 4). All sequences that were used in our study with the indication of accession numbers and comments on sequence status are also provided (Table S1).

Discussion
The classification of criconematid species has until now been based on morphology alone (especially cuticular markings) and is widely considered to be volatile 1,9 . Although Subbotin et al. 10 observed the monophyly of the genera Mesocriconema, Hemicriconemoides, and Criconema in the suborder Criconematina based on D2-D3 data, our analysis based on more recent 18S, D2-D3, and COI sequences indicated none of these genera as being monophyletic. Likewise, the phylogenetic analysis of Powers et al. 11 , based on 18S sequences, showed that Lobocriconema is a monophyletic group and Criconemoides is paraphyletic. However, our 18S, D2-D3, and COI tree topologies indicated that Lobocriconema and Criconemoides are also both polyphyletic groups (Figs. 1, 2, 3). Furthermore, the SH and AU tests based on 18S, D2-D3, and/or COI datasets rejected the monophyly hypothesis of eight genera in Criconematidae, i.e. Criconema, Ogma, Crossonema, Discocriconemella, Hemicriconemoides, Criconemoides, Mesocriconema, and Lobocriconema. Thus, based on current and former molecular analyses, none of the generic groupings within the family Criconematidae can be supported, except for those genera with limited available sequences on GenBank (Bakernema, Nothocriconemoides and Neolobocriconema). Our updated molecular analyses suggest that key morphological characters used in the classification of Criconematidae have www.nature.com/scientificreports/ resulted from convergent evolution. For example, the species of genus Hemicriconemoides with double cuticle did not form a monophyletic group; the large labial disc in Discocriconemella appears to have evolved at least three times independently, in agreement with previous studies 11,39 ; the very coarse annuli typical for Lobocriconema appeared in two distant clades; species belonging to Criconemoides (without cuticular outgrowths) appear in three different clades; the genera Criconema (cuticle with continuous fringe or longitudinal rows) and Ogma (cuticle www.nature.com/scientificreports/ with numerous appendages, arranged in 6-26 longitudinal rows) appear in at least two different phylogenetic conditions (Figs. 2, 3, 4, 6).
In this study, we also revealed the remarkable molecular variations in several criconematid species, a case in point being our population of C. myungsugae. This nematode population agrees morphologically with the type population and other descriptions. Although 18S, D2-D3, and COI analyses indicated its close relation with other populations of C. myungsugae, remarkable molecular variations between sequences of C. myungsugae can be observed. Intrapopulation variations of C. myungsugae from Vietnam for 18S, D2-D3, and COI sequences Table 3. Comparing the number of established species and molecular species-delimitation results according to different species-delimitation methods of Criconematidae with sequence representatives in GenBank.   www.nature.com/scientificreports/ were respectively 0.9%, 3.7%, and 0%, and they differed by 0.5-1.1%, 2.3-5.1%, and 10.9-11% respectively to the sequences of C. myungsugae from GenBank. Criconemoides myungsugae from Vietnam was, therefore, also appointed as a distinct species (or may even be appointed as several species) by several of the molecular speciesdelimitation approaches that were employed. Similarly, high molecular variations were also recorded among 18S, D2-D3, and COI sequences of D. hengsungica (0.3%, 4.3%, and 12.3%, respectively) that also resulted in putative species splitting, according to several of the species-delimitations methods used. Other authors 35,36,40 considered such molecular differences sufficient to establish the description of new species. However, given the high intraspecific/intrapopulation variations observed in this study, the distinction between intra-and interspecific variations (the essence of molecular species-delimitations) is not unequivocal. This is also reflected in the contradictions between the used species-delimitation methods, with only ABGD results (ABGD1 and ABGD2) appointing the respective populations of C. myungsugae and D. hengsungica as conspecific according to 18S and D2-D3 analyses (Figs. 2, 3). It is notable that virtually all the molecular species-delimitation approaches used in this study resulted in a considerably larger number of putative species compared to the number of established species (Tables 3, 4). These results agree with other studies indicating that tree-based and distance-based species-delimitation methods tend to recover a higher number of phylogenetic lineages than the number of species originally considered 29,41,42 . Conversely, the ABGD2 and bPTP output appears to be over-conservative when delimitating several Crossonema, Criconema, and Ogma species. For example, the 18S sequences of five species including Crossonema fimbriatum, C. menzeli, Ogma cobbi, Ogma murrayi, and Ogma seymouri were delineated as a single species. However, the number of putative species detected according to the ABGD2 output remarkably well agreed with the number of established species (after taking the presented suggestions into account), i.e. an identical, 5 less, and 6 additional species in comparison, respectively for the 18S, D2-D3, and COI dataset (Table 3, Figs. 2, 3,  4). Nonetheless, despite this attractively convenient-looking match, we cannot simply champion ABGD as the preferred method for molecular species delineation, given the highly inconsistent ABGD outputs (i.e. ABGD1 vs ABGD2). We also concur with Hofmann et al. 29 and Prevot et al. 43 that to avoid over-or underestimation of molecular species delimitation, a combination of tree-based and distance-based methods should be employed to ensure a more stable taxonomic interpretation. Taken all together, the selection of a single species-delimitation method is not straightforward, and thus, molecular species-delimitation methods should be considered as one of the techniques to be used in comprehensive polyphasic taxonomy. Furthermore, this study also clearly revealed that, for criconematid species, 40 sequences belonging to 14 different species on GenBank have been mislabeled, unlabeled, associated with misidentified sequences, or need to be reviewed, a finding that agrees with previous studies 31,44 . Therefore, both the new species descriptions as well as records of known species must be validated by polyphasic studies which combine analyses of both morphological and molecular approaches. This represents the clearest way forward in order to avoid confusion and to advance nematode taxonomy in general 31,34,41,[45][46][47][48] .

Conclusions
Phylogenetic analyses, supported by tree topology tests, based on different gene regions confirmed that virtually all studied criconematid genera (except for those with limited available sequences in GenBank) are not monophyletic groups. Key morphological characters used in the classification of Criconematidae are likely to be the consequence of convergent evolution. This study provides the first report of Criconemoides myungsugae and Discocriconemella hensungica associated with Vietnamese ginseng, one of the most precious and rare ginseng varieties in the world. Besides, our molecular analyses also revealed the high molecular intraspecific variations, www.nature.com/scientificreports/ with our nematode populations as cases in point, in many criconematid species that were considered as enough to establish new cryptic species by some nematologists. The combination of different tree-and distance-based molecular species delimitation methods used in our study have helped in the declamation of molecular species boundary more stably. Our polyphasic study also indicated a number of sequences from GenBank must be either revised or reconsidered.